Breast cancer stem cells have been identified as CD44?/low breast tumor cells that exclusively retain the ability to form new tumors in mouse models. Cancer stem cells can arise from mutations in normal stem cells directly, or from mutations in progenitor cells that lead to the regaining of stem cell property. Although normally down-regulated during mammary epithelial differentiation, CD44 is expressed in up to 85% human breast cancers and the ectopic expression of CD44 has been shown to promote metastasis. It is possible that the deregulation of CD44 in breast stem cell is associated with tumorigenic transformation due to mutations in pathways that regulate CD44 expression. Therefore it is extremely important to dissect the molecular mechanism(s) that regulates CD44 expression in breast stem cells and controls CD44 down-regulation during stem cell differentiation. Gene expression can be controlled by genetic elements (e.g., cis-regulators, i.e., non- coding DNA sequences that control cell type-specific expression/suppression of genes), in addition to epigenetic controls such as DNA methylation. Due to the highly complex nature of the genome, finding cis-regulators, which determine cell type-specific (stem cells versus differentiated cell) gene expression, in vertebrates remains a difficult task. However, the fact that many cis-regulators are often evolutionarily conserved across species provides a basis for the identification of these cis-regulators using comparative genomic method. Our approach employing in silico predication and "wet lab" verification methods to identify crucial cis-regulators and trans-acting factors of the CD44 gene is highly innovative. Evolutionarily conserved non-coding regions for the prediction of cis-regulators greatly reduced false positives, hence, work load for experimental verification. It provides an integrative way to decode the complicated transcriptional regulation of CD44. Understanding regulatory mechanisms of CD44 expression could ultimately lead to the identification of pathways that could be mutated during the tumorigenic transformation of breast stem cells or cells further down the differentiation hierarchy and thus may provide new therapeutic targets for treatment of breast cancer. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to dissect molecular mechanisms that regulate the expression of the breast cancer stem cell marker CD44 .Understanding regulatory mechanisms of CD44 expression could ultimately lead to the identification of pathways that could be mutated during tumorigenic transformation of breast stem cells or cells further down the differentiation hierarchy and thus provide novel therapeutic targets for treatment of breast cancer, aimed directly at the origin of the tumor.